5c8 Antigen

ABSTRACT

This invention provides a monoclonal antibody which specifically recognizes and forms a complex with a protein located on the surface of activated T cells and thereby inhibits T cell activation of B cells. This invention also provides the monoclonal antibody 5c8 (ATCC Accession No. ______).  
     This invention provides a human CD4 −  T cell leukemia cell line designated D1.1 (ATCC Accession No. ______) capable of constitutively providing contact-dependent helper function to B cells. This invention also provides an isolated protein from the surface of activated T cells, wherein the protein is necessary for T cell activation of B cells. This invention further provides an isolated, soluble protein from the surface of activated T cells, wherein the protein is necessary for T cell activation of B cells.

The invention described herein was made in the course of work undergrant Nos. PO1-AI-26886, RO-1-AI-14969 and Immunology Training GrantAI-07132 from the National Institutes of Health. The United Statesgovernment therefore has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byArabic numerals within parenthesis. Full citations for thesepublications may be found at the end of the specification, immediatelypreceding the claims. The disclosures of these publications are herebyincorporated by refernce into this application in order to more fulldescribe the state of the art as known skilled therein as of the date ofthe invention described and claimed herein.

In a contact-dependent process termed “T cell helper function,” CD4⁺ Tlymphocytes direct the activation and differentiation of B lymphocytesand thereby regulate the humoral immune response by modulating thespecificity, secretion and isotype-encoded functions of antibodymolecules (1-8). The T cell surface molecules that mediate thecontact-dependent elements of T cell helper function are not yet fullyknown (9).

The process by which T cells help B cells to differentiate has beendivided into two distinct phases: the inductive and effector phases (10,11). In the inductive phase, resting T cells contact antigen-primed Bcells and this association allows clonotypic. T cell receptor (TCR)-CD4complexes to interact with Ia/Ag complexes on B cells (5, 12-19).TCR/CD4 recognition of Ia/Ag results in the formation of stable T-Bcognate pairs and bidirectional T and B cell activation (20-26). In theeffector phase, activated T cells drive B cell differentiation bysecreting lymphokines (27-30) and by contact-dependent stimuli (24,31-38), both of which are required for T cells to drive small, resting Bcells to terminally differentiate into Ig secreting cells (31, 39-42).

Although the inductive phase of T cell help is Ag-dependent andMHC-restricted (5, 12-18, 40), the effector phase of T cell helperfunction can be Ag-independent and MHC-nonrestricted (31, 34, 36, 40,43-50). An additional contrasting feature is that the inductive phase ofT cell help often requires CD4 molecules and is inhibited by anti-CD4mAb (19), whereas helper effector function does not require CD4molecules (51) and is not inhibited by anti-CD4 mAbs (33, 34, 36, 49).The nonspecific helper effector function is believed to be focused onspecific B cell targets by the localized nature of the T-B cellinteractions with antigen specific, cognate pairs (0.25, 26, 52).

Although terminal B cell differentiation requires both contact- andlymphokine-mediated stimuli from T cells, intermediate stages of B celldifferentiation can be induced by activated T cell surfaces in theabsence of secreted factors (32, 33, 53-56). These intermediate effectson B cells include induction of surface CD23 expression (32, 35, 57),enzymes associated with cell cycle progression (37) and responsivenessto lymphokines (24, 37, 4.9, 54-56). Although the activation-induced Tcell surface molecules that direct B cell activation have not beenidentified, functional studies have characterized some features of theirinduction and biochemistry. First, T cells acquire the ability tostimulate B cells 4-8 h following activation (38, 49).

Second, the B cell stimulatory activity associated with the surfaces ofactivated T cells is preserved on paraformaldehyde fixed cells (24, 32,37, 49, 56) and on purified membrane fragments (33, 53-55). Third, the Bcell stimulatory activity is sensitive to protease treatment (24, 53,54). Fourth, the process of acquiring these surface active structuresfollowing T cell activation is inhibited by cyclohexamide (49, 54).Although these studies strongly suggest the existence ofactivation-induced T cell surface proteins that deliver contactdependent stimuli to B cells, the molecular identities of suchstructures have not yet been described.

SUMMARY OF THE INVENTION

This invention provides a monoclonal antibody which specificallyrecognizes and forms a complex with a protein located on the surface ofactivated T cells and thereby inhibits T cell activation of B cells.This invention also provides the monoclonal antibody 5c8 (ATCC AccessionNo.

This invention provides a human CD4⁻ T cell leukemia cell linedesignated D1.1 (ATCC Accession No. ______) capable of constitutivelyproviding contact-dependent helper function to B cells. This inventionalso provides an isolated protein from the surface of activated T cells,wherein the protein is necessary for T cell activation of B cells. Thisinvention further provides an isolated, soluble protein from the surfaceof activated T cells, wherein the protein is necessary for T cellactivation of B cells.

Monoclonal antibody 5c8 and a human CD4⁻ T-Cell line., designated, D1.1have been deposited with the American Type Culture Collection, 12301Parklawn Drive, Rockville Md., 20852, U.S.A, pursuant to the provisionsof the Budapest Treaty on the International Recognition of theMicroorganism Deposit for the Purposes of Patent Procedure and have beenaccorded ATCC Accession Nos. ______ and respectively.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Cell surface phenotype of CD⁻ Jurkat D1.1. Shown arefluorescence histogram (FACS) analyses of CD4⁻ Jurkat D1.1 and CD4⁺B2.7. The Y axis represents number of cells and the X axis representsrelative fluorescence intensity. The mAb used are: OKT3(anti-CD3),OKT4(anti-CD4), W6/32(anti-MHC I). “Control” represents the backgroundstaining in the absence of added primary mAb.

FIG. 2. Jurkat D1.1 induces CD23 expression on resting B lymphocytes.Shown are two-color FACS analyses of adherence depleted, high density Bcells after 24 h of culture alone (media) or with CD4⁻ Jurkat (D1.1) orCD4⁺ Jurkat (B2.7) by using anti-IgM-FITC or anti-CD20 (Leu-16)—FITC (onthe X-axis) and anti-CD23-PE (on the Y-axis) (Becton-Dickinson). Thenumbers shown in the upper right hand corner of each histogramrepresents the percentage of all gated cells that express bothmolecules. The population of B cells cultured with Jurkat D1.1 expressedCD23 on 66% of IgM⁺ cells and 69% of CD20⁺ cells compared with B cellscultured with B2.7 (16% of IgM⁺ and 16% of CD20⁺ cells). In theexperiment shown, single color FACS showed the population of small, highdensity B cells to be 2% CD3(OKT3)⁺, 84% IgM⁺, 84% CR2(HB-5)⁺, and 87%CD20(Leu-16)⁺.

FIG. 3. Dose response of D1.1-induced CD23 expression. Shown are thepercentage of IgM⁺ cells that express CD23 after 24 h culture withvarying ratios of D1.1 or B2.7 cells or cell supernatants. Experimentalconditions and two-color FACS analysis were as described in FIG. 2,legend except that the ratio of Jurkats added to 2×10⁵ cells was variedas shown. Supernatants were obtained 48 h after 1×10⁵ D1.1 or B2.7 cellswere cultured in 1 ml of Iscove's modified Dulbecco medium/10% FCS andwere passed through 0.2-mμ filters before addition to the B cells. Thebackground level (B cells alone) of CD23 expression of IgM cells was12%. The B cell population was 65% IgM⁺ in this experiment.

FIG. 4 FIG. 4 shows that Jurkat D1.1 induces B cell proliferation in thepresence of PHA. Shown is [H³] thymidine uptake of B cells cultured withmitomycin-C-treated Jurkat cells in the presence of the indicatedcombination of rIL-2(25 U/ml), rIL-4(25 U/ml)_(g) PHA (5 ug/ml), orcontrol media. Error bars represent standard deviation of the means oftriplicate cultures.

FIG. 5. Jurkat D1.1 induces B cell differentiation into Ig secretingcells. A. Number of plaque-forming colonies per 10⁵ B cells induced byindicated ratios of Jurkat D1.1 or B2.7 to B cells in the presence ofabsence of PWM B. IgG in supernatants from the same experiment as in1A). E??? cells are E rosette-depleted, adherence-depleted, high densityPercoll population that is predominantly B cells. E?? cells are Erosette-positive, resting T cells treated with mitomycin-C. Measurementof Ig was performed by quantitative sandwich ELISA and error barsrepresent calculated standard deviation based on standard curves.

FIG. 6. rIL-4 but not D1.1 increased B cell sigM expression. Shown aretwo-color FACS analyses resulting from experiments similar to those inFIG. 3. The concentration of anti-IL-4 shown is 1.25 μg/ml and theconcentration of rIL-4.50 U/ml. The median channel fluorescence of IgMis shown on the right column.

FIG. 7. Binding of mAb 5c8 to Jurkat D1.1 cells. Shown are fluorescencehistogram (FACS) analyses of CD4⁻ Jurkat D1.1 and CD4⁺ Jurkat B2.7cells. The Y axis represents number of cells and the X axis representsrelative fluorescence intensity. The mAbs used are: OKT2(anti-CD3),OKT4(anti-CD4), OKT8(anti-CD8), W6/32(anti-MHC I) and mAb 5c8. FITCrepresents the background staining of an isotype matched control mAb.

FIG. 8. mAb 5c8 inhibits Jurkat. D1.1 induced CD23 expression by BLymphocytes. Shown are two color FACS analyses of adherence depleted,high density B cells after 24 h of culture alone or with the B2.7 orD1.1 Jurkat clones using anti-IgM-FITC (the X axis) and anti-CD23-PE (onthe Y axis). The number in the upper right hand corner of the FACStracings represents the percentage of IgM⁺ cells that expressed CD23.The mAb W6/32 was present at 1 μg/ml, the mAb 5c8 at a 1:200 dilution ofhybridoma supernatant. The murine IgG2a mAb W6/32 recognizes amonomorphic determinant on Class I MHC molecules.

FIG. 9. SDS/PAGE analysis of surface proteins immunoprecipitated by mAb5c8 and control mAbs. Shown are autoradiograms of immunoprecipitateswith m Ab 5c8 or control mAbs from cell lysates of surface iodinatedJurkat D1.1 or Jurkat B2.7 cells that were separated on 12.5%polyacrylamide in the presence (reduced, R) or absence (non-reduced, NR)or 2-ME. mAbs shown are anti-CD28 (KOLT-4) and anti-MHC Class I (W6/32).MW markers represent the migration of pre-labelled standards. NMS:normal mouse serum.

FIG. 10. Effects of T cell activation and metabolic inhibitors on theexpression of 5c8 antigen on activated T cells. Shown are FACShistograms of resting and activated T cells using mAb 5c8 or anti-CD69as indicated. T cell activation was marked by PMA (10 ng/ml) and PHA (10μg/ml) for 5 h performed in the presence of actinomycin D (10 μM) orcyclohexamide (100 μM).

FIG. 11. Kinetics of expression of 5c8 on isolated CD4⁺ or CD8⁺ T cellsubsets. Shown are fluorescence histograms of (a, b, c) CD4⁺ cells or(d, e, f) CD8⁺ cells at the indicated time points after freshly purifiedT cell subsets were activated with PHA (10 μg/ml) and PMA (10 ng/ml).Solid line: 5c8 binding; dashed line: IgG2a control; and dotted line:anti-CD69.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a monoclonal antibody which specificallyrecognizes and forms a complex with a protein located on the surface ofactivated T cells, thereby inhibiting T cell activation of B cells.Activated T cells are found normally only in the germinal centers of ananimal's lymph nodes. However, activated T cells are found in theperipheral blood of animals suffering from T cell tumors, e.g., T cellleukemias and lymphomas.

The monoclonal antibody described and claimed herein binds to T cellswhich are interacting with B cells in the germinal centers of lymphnodes and not to other T cells. Monoclonal antibodies known to thoseskilled in the art to specifically recognize and bind to proteins on thesurface of T cells and thereby inhibit the activation of B cells, e.g.,anti-CD28 monoclonal antibody and anti-LFA-1 monoclonal antibody, do notdistinguish activated T cells.

For the purposes of this invention, “activated T cells” are T cellscapable of providing T cell helper function to resting B cells. For thepurposes of this invention, “germinal centers of lymph nodes” are theareas in lymph nodes where T cells provide T cell helper function to Bcells.

For the purposes of this invention a “monoclonal antibody” is anantibody produced by a hybridoma cell. Methods of making monoclonalantibody-synthesizing hybridoma cells are well known to those skilled inthe art, e.g, by the fusion of an antibody producing B lymphocyte withan immortalized B-lymphocyte cell line.

In one embodiment of this invention, the B cells are resting B cells. Inanother embodiment of this invention, the B cells are primed B cells.For the purposes of this invention, “resting” B cells are unactivated Bcells, i.e., undifferentiated B cells which do not synthesize antibodymolecules. For the purposes of this invention, “primed” B cells are Bcells which have been contacted with antigen and have thereby beenpartially activated, but which do not yet synthesize antibody molecules.

In one embodiment of this invention, the monoclonal antibody is a murinemonoclonal antibody. In another embodiment of this invention, themonoclonal antibody is a chimaeric monoclonal antibody. In still anotherembodiment of this invention, the monoclonal antibody is a humanizedmonoclonal antibody. However, in the preferred embodiment of thisinvention, the monoclonal antibody is a human monoclonal antibody.

For the purposes of this invention, a “chimaeric” monoclonal antibody isa murine monoclonal antibody comprising constant region fragments(F_(c)) from a different animal. In a preferred embodiment of thisinvention, the chimaeric monoclonal antibody comprises human F_(c) andmurine F_(c). For the purposes of this invention, a “humanized”monoclonal antibody is a murine monoclonal antibody in which humanprotein sequences have been substituted for all the murine proteinsequences except for the murine complement determining regions (CDR) ofboth the light and heavy chains.

In one embodiment of this invention, the monoclonal antibody is directedto the protein to which the monoclonal antibody 5c8 (ATCC Accession No.______) is directed. In another embodiment of this invention, themonoclonal antibody is directed to the epitope to which the monoclonalantibody 5c8 (ATCC Accession No. ______) is directed. In still anotherembodiment of this invention, the monoclonal antibody is the monoclonalantibody 5c8.

In one of this invention, the monoclonal antibody is labelled with adetectable marker, for example, a radioactive isotope, enzyme, dye orbiotin. In another embodiment of this invention, the monoclonal antibodyis conjugated to a therapeutic agent, for example, a radioisotope,toxin, toxoid or chemotherapeutic agent. In still another embodiment ofthis invention, the monoclonal antibody is conjugated to an imagingagent for example, a radioisotope.

This invention provides a pharmaceutical composition comprising themonoclonal antibody and a pharmaceutically acceptable carrier. For thepurposes of this invention “pharmaceutically acceptable carriers” meansany of the standrad pharmaceutical carriers. Examples include, but arenot limited to, physiological saline, phosphate buffered salinecontaining Polysorb 80 or water.

The monoclonal antibodies described and claimed herein are useful forisolating the proteins to which the monoclonal antibodies bind. Thewmonoclonal antibodies are also valuable in new and useful methods for:inhibiting the immune response in an animal; imaging T cell tumors in ananimal; detecting the presence of a T cell tumor in an animal;determining whether an animal harbors a T cell tumor which comprises;inhibiting the proliferation of T cell tumor cells in an animalsuffering from a T cell cancer; and inhibiting viral infection of the Tcells of an animal.

This invention provides an isolated nucleic acid molecule encoding thelight chain protein of the monoclonal antibody. In one embodiment ofthis invention, the nucleic acid molecule is a DNA molecule. Preferably,the DNA molecule is a cDNA molecule.

The nucleic acid sequences described and claimed herein are useful forgenerating new viral and circular plasmid vectors described below. Thenucleic acid molecules are also valuable in a new and useful method ofgene therapy, i.e., by stably transforming cells isolated from an animalwith the nucleic acid molecules and then readministering the stablytransformed cells to the animal. Methods of isolating cells include anyof the standard methods of withdrawing cells from an animal. Suitableisolated cells include, but are not limited to, bone marrow cells.Methods of readministering cells include any of the standard methods ofreadministering cells to an animal.

This invention provides a gene transfer vector, for example a plasmid ora viral vector, comprising a nucleic acid molecule encoding the lightchain protein of the monoclonal antibody operably linked to a promoterof RNA transcription. This invention also provides a gene transfervector, for example a plasmid or a viral vector, comprising a nucleicacid molecule encoding the heavy chain protein of the monoclonalantibody operably linked to a promoter of RNA transcription.

The gene transfer vectors described and claimed herein are valuable asproducts useful for generating stably transformed eukaryotic host cells,and thereby in new and useful methods of growing such host cells underconditions suitable for the production of a protein.

This invention provides a host vector system comprising the genetransfer vectors described and claimed herein in a suitable host cell.In one embodiment of this invention, the suitable host cell is a stablytransformed eukaryotic cell, for example a stably transformed yeast or amammalian cell. In the preferred embodiment of this invention, thestably transformed eukaryotic cell is a stably transformed mammaliancell.

The host vector system described and claimed herein is valuable in a newand useful method for the synthesis of a monoclonal antibody, comprisinggrowing the host vector system under conditions suitable for theproduction of the monoclonal antibody.

This invention provides a hybridoma cell producing a monoclonal antibodyof this invention. Preferably, the hybridoma cell is the hybridoma cellproducing the monoclonal antibody 5c8 (ATCC Accession No. ______). Forthe purposes of this invention, a “hybridoma cell” is a cell formed bythe fusion of an immortalized cell and an antibody-producing cell,thereby forming a cell which makes a monoclonal antibody.

This invention provides a CD4 human T cell leukemia cell line designatedD1.1 (ATCC Accession No. ______) capable of constitutively providingcontact-dependent helping function to B cells. In one embodiment of thisinvention, the B cells are resting B cells. In another embodiment ofthis invention, the B cells are primed B cells.

The cell line described and claimed herein is valuable as a source ofthe isolated activated T cell surface protein, which is valuable for theinformation it provides concerning the nucleotide sequences which encodeit. The nucleotide sequences are valuable in a new and useful method ofproducing the soluble activated T cell surface protein described andclaimed herein. The cell line is also valuable in new and useful methodsfor immunizing an animal against a protein antigen and for screeningpharmaceutical compounds for their ability to inhibit T cell activationof B cells.

This invention provides an isolated protein from the surface ofactivated T cells that is necessary for T cell activation of B cells. Inone embodiment of this invention, the B cells are resting B cells. Inanother embodiment of this invention, the B cells are primed B cells.Preferably, the isolated protein is the protein to which the monoclonalantibody 5c8 (ATCC Accession No. ______) binds.

This invention provides an isolated nucleic acid molecule encoding the Tcell surface protein. In one embodiment of this invention, the nucleicacid molecule is a DNA molecule. Preferably, the DNA molecule is a cDNAmolecule. The nucleic acid molecules are valuable as products forgenerating new viral and circular plasmid vectors described below. Thenucleic acid molecules are also valuable in a new and useful method ofgene therapy, i.e., by stably transforming cells isolated from an animalwith the nucleic acid molecules and then readministering the stablytransformed cells to the animal. Methods of isolating cells include anyof the standard methods of withdrawing cells from an animal. Suitableisolated cells include, but are not limited to, bone marrow cells.Methods of readministering cells include any of the standard methods ofreadministering cells to an animal.

This invention also provides a gene transfer vector, for example aplasmid or a viral vector, comprising the isolated nucleic acid moleculeencoding the actiavted T cell surface protein.

The gene transfer vectors described and claimed herein are valuable asproducts useful for generating stably transformed eukaryotic host cells,and thereby in new and useful methods of growing such host cells underconditions suitable for the production of a protein.

This invention further provides a host vector system comprising the genetransfer vector in a suitable host cell. In one embodiment of thisinvention, the suitable host cell is a stably transformed eukaryoticcell, for example, a stably transformed eukaryotic yeast or a mammaliancell. Preferably, the stably transformed cell is a stably transformedmammalian cell.

The host vector system is valuable as a product useful for the largescale synthesis of the activated T cell surface protein by growing thehost vector system under conditions suitable for the production ofprotein. Thus, a method of producing the activated T cell surfaceprotein is also provided. This invention further provides the proteinproduced by this method.

This invention provides an isolated, soluble protein from the surface ofactivated T cells necessary for T cell activation of B cells. In oneembodiment of this invention, the B cells are resting B cells. Inanother embodiment of this invention, the B cells are primed B cells.

For the purposes of this invention, a “soluble protein” is a proteinfree of cell membranes and other cellular components. Preferably, thesoluble protein is the protein to which the monoclonal antibody 5c8(ATCC Accession No. ______) binds. In one embodiment of this invention,the soluble protein is labelled with a detectable marker, for example, aradioactive isotope, enzyme, dye or biotin. The soluble protein isvaluable as a product for making a new and useful pharmaceuticalcomposition.

Thus, a pharmaceutical composition comprising the soluble protein and apharmaceutically acceptable carrier is also provided. “Pharmaceuticallyacceptable carriers” means any of the standard pharmaceuticallyacceptable carriers. Examples include, but are not limited to, phosphatebuffered saline, physiological saline, water and emulsions, such asoil/water emulsions.

This invention provides an isolated nucleic acid molecule encoding thesoluble protein. In one embodiment of this invention, the nucleic acidmolecule is a DNA molecule. Preferably, the DNA molecule is a cDNAmolecule.

The nucleic acid sequences described and claimed herein are useful forgenerating new viral and circular plasmid vectors described below. Thenucleic acid molecules are also valuable in a new and useful method ofgene therapy, i.e., by stably transforming cells isolated from an animalwith the nucleic acid molecules and then readministering the stablytransformed cells to the animal. Methods of isolating cells include anyof the standard methods of withdrawing cells from an animal. Suitableisolated cells include, but are not limited to, bone marrow cells.Methods of readministering cells include any of the standard methods ofreadministering cells to an animal.

This invention also provides a gene transfer vector, for example, aplasmid vector or a viral vector, comprising the isolated nucleic acidmolecule operably linked to a promoter of RNA transcription.

The gene transfer vectors described and claimed herein are valuable asproducts useful for generating stably transformed eukaryotic host cells,and thereby in new and useful methods of growing such host cells underconditions suitable for the production of a protein.

This invention further provides a host vector system comprising the genetransfer vector in a suitable host cell. In one embodiment of thisinvention, the suitable host cell is a stably transformed eukaryoticcell, for example, a stably transformed eukaryotic yeast or mammaliancell. Preferably, the stably transformed cell is a stably transformedmammalian cell.

The host vector system is valuable as a product useful for the largescale synthesis of the soluble activated T cell surface protein bygrowing the host vector system under conditions suitable for theproduction of protein and recovering the protein so produced. Thus, amethod of producing the soluble protein is also provided. This inventionfurther provides the soluble protein produced by this method.

This invention provides a method of inhibiting B cell activation in ananimal which comprises administering to the animal an effectiveinhibiting amount of a pharmaceutical composition comprising themonoclonal antibody which specifically recognizes the activated T cellsurface protein and a pharmaceutically acceptable carrier. For thepurposes of this invention, an “effective inhibiting amount” of apharmaceutical composition is any amount of the pharmaceuticalcomposition which is effective to bind to a protein on the surface ofactivated T cells and thereby inhibit T cell activation of B cells. Inone embodiment of this invention, the B cells are resting B cells. Inanother embodiment of this invention, the B cells are primed B cells.

Methods of determining an “effective amount” are well known to thoseskilled in the art and will depend upon factors including, but notlimited to, the type of animal involved and the animal's body weight. Inone embodiment of this invention, the animal is a mammal, for example amouse or a human. Preferably, the mammal is a human.

For the purposes of this invention, “administration” means any of thestandard methods of administering a pharmaceutical composition known tothose skilled in the art. Examples include, but are not limited to,intravenous, intraperitoneal or intramuscular administration.

The method of inhibiting B cell activation is valuable in a new anduseful method for inhibiting the immune response of an animal. In oneembodiment of this invention, the animal is a mammal, for example amouse or a human. Preferably, the mammal is a human.

In one embodiment of this invention, inhibiting the immune response ofan animal is valuable as a method of inhibiting the rejection by theanimal of a transplant organ, for example, a heart, kidney or liver.

In another embodiment of this invention, inhibiting the immune responseof an animal is valuable as a method of inhibiting the autoimmuneresponse in an animal suffering from autoimmune disease. Examples ofautoimmune diseases include, but are not limited to, rheumatoidarthritis, Myasthenia gravis, systemeic lupus erythrematosus, Graves'disease, idiopathic thrombocytopenia purpura, hemolytic anemia, diabetesmellitus and drug-induced autoimmune diseases, e.g., drug-induced lupus.

In still another embodiment of this invention, inhibiting the immuneresponse in an animal is valuable as a method of inhibiting allergicresponses, e.g., hay fever or an allergy to penicillin, in the animal.

This invention provides a method of imaging T cell tumors, e.g., T cellleukemias or lymphomas, in a patient which comprises: administering tothe patient an effective imaging amount of a pharmaceutical compositioncomprising the monoclonal antibody which specifically recognizes theactivated T cell surface protein, conjugated to an imaging agent, underconditions permitting the formation of a complex between the monoclonalantibody and a protein on the surface of the tumor cells; and imagingany monoclonal antibody/protein complex formed, thereby imaging any Tcell tumors in the patient. Preferably, the patient is a human patient.

The T cell surface protein is found in animals free of tumors only onthe surface of activated T cells, i.e., those T cells providingcontact-dependent helper function to B cells in the germinal centers oflymph nodes. However, the protein is found on the surface of T celltumor cells circulating in the blood of the animal.

“Administering” means any of the standard methods of administering aphramaceutical composition known to those skilled in the art. Examplesinclude, but are not limited to intravenous, intramuscular orintraperitoneal administration. Methods of detecting the formation ofmonoclonal antibody/protein complexes, e.g., by exposure of x-ray film,are well known to those skilled in the art.

An “effective imaging amount” of the pharmaceutival composition is anyamount effective for the formation of complexes between the monoclonalantibody and a cell surface protein, such that the complexes can beimaged. Methods of determining an “effective imaging amount” are wellknown to those skilled in the art and depend upon factors including, butnot limited to the type of animal involved, the size of the animal andthe imaging agent used. In one embodiment of this invention, the imagingagent is a radioisotope.

This invention provides a method of detecting the presence of a T celltumor, e.g., a T cell leukemia or lymphoma, in an animal whichcomprises: administering to the animal an amount of a pharmaceuticalcomposition comprising a monoclonal antibody bound to an detectablemarker effective to bind to a protein on the surface of T cell tumorcells under conditions permitting the formation of complexes between themonoclonal antibody and the protein; clearing any unbound imaging agentfrom the animal; and detecting the presence of any monoclonalantibody/protein complex so formed, the presence of such complexindicating the presence of T cell tumor cells in the animal. In oneembodiment of this invention, the animal is a mammal, e.g., a mouse or ahuman. Preferably, the mammal is a human.

“Administering” means any of the standard methods of administering aphramaceutical composition known to those skilled in the art. Examplesinclude, but are not limited to intravenous, intramuscular orintraperitoneal administration. Methods of detecting the formation ofmonoclonal antibody/protein complexes, e.g., by exposure of x-ray filmor microscopic examination, are well known to those skilled in the art.

An “effective amount” of the pharmaceutical composition is any amount ofthe pharmaceutical composition effective to detect the presence of a Tcell tumor in the animal. Methods of determining an “effective amount”are well known to those skilled in the art and depend upon a number offactors including, but not limited to: the type of animal involved, thesize of the blood sample contacted and the detectable marker used. Inone embodiment of this invention, the detectable marker is aradiosotope, enzyme, dye or biotin.

This invention provides a method of determining whether an animalharbors a T cell tumor, e.g., a T cell leukemia or lymphoma, whichcomprises: isolating a sample of blood from the animal; contacting saidsample with an amount of pharmaceutical composition comprising amonoclonal antibody, wherein the monoclonal antibody is labelled with adetectable marker, effective to bind to a soluble protein underconditions permitting the formation of a complex between the monoclonalantibody and the protein; and detecting the presence of any monoclonalantibody/protein complex so formed, the presence of such complexindicating the presence of T cell tumor cells in the patient.

The method provided by this invention is valuable as a new and usefulmethod of detecting the presence of T cell tumor cells in the blood ofan animal before the presence of the tumor cells themselves can bedetected. The method provided by this invention is also valuable as anew and useful method for determining the effectiveness of the treatmentof an animal with an anti-T cell tumor drug, i.e., by determining thelevel of soluble protein in the blood of the animal, such level beingindicative of the effectiveness of the treatment.

It is well known to those skilled in the art that the blood of patientssuffering from T cell tumors contains soluble proteins, e.g., the tacantigen, shed from the surface of T cell tumor cells. Thus, the presenceof soluble T cell surface proteins in the blood of an animal isindicative of the presence of T cell tumors in the animal.

For the purposes of this invention, a “soluble protein” is a proteinfree of cell membranes and other cellular components. In the preferredembodiment of this invention, the soluble protein is the protein towhich thwe monoclonal antibody 5c8 (ATCC Accession No. ______) binds.

“Isolating” blood from an animal means any of the generally acceptablemethods of withdrawing blood and immediately placing the blood into areceptacle containing an anticoagulaht, e.g., heparin, EDTA or citrate.Methods of detecting monoclonal antibody/protein complexes are wellknown to those skilled in the art. Examples include, but are not limitedto, exposure of x-ray film and ELISA.

An “effective amount” of the pharmaceutical composition is any amount ofthe pharmaceutical composition effective to detect the presence of thesoluble protein in the blood of the animal. Methods of determining an“effective amount” are well known to those skilled in the art and dependupon a number of factors including, but not limited to: the type ofanimal involved, the size of the blood sample contacted and thedetectable marker used. In one embodiment of this invention, thedetectable marker is a radiosotope, enzyme, dye or biotin.

In one embodiment of this invention, the animal is a mammal, e.g., amouse or a human. Preferably, the mammal is a human.

This invention provides a method of inhibiting the proliferation of Tcell tumor cells, in an animal suffering from a T cell cancer, e.g., a Tcell leukemia or lymphoma, which comprises administering to the patientan amount of the pharmaceutical composition, comprising a monoclonalantibody conjugated to a therapeutic agent, effective to inhibit theproliferation of T cell tumor cells. In one embodiment of thisinvention, the animal is a mammal, e.g., a mouse or a human. Preferably,the mammal is a human.

“Administering” means any of the standard methods of administering aphramaceutical composition known to those skilled in the art. Examplesinclude, but are not limited to intravenous, intramuscular orintraperitoneal administration.

An “effective amount” of the pharmaceutical composition is any amount ofthe pharmaceutical composition effective to inhibit the proliferation ofT cell tumor cells. Methods of determining an “effective amount” arewell known to those skilled in the art and depend upon factorsincluding, but not limited to: the type of animal involved, the size ofthe animal and the therapeutic agent used. In one embodiment of thisinvention, the therapeutic agent is a radioisotope, toxin, toxoid orchemotherapeutic agent.

This invention provides a method of inhibiting viral infection of the Tcells of an animal by the HTLV I virus comprising administering to theanimal an amount of a pharmaceutical composition, comprising amonoclonal antibody which specifically recognizes a protein on thesurface of activated T cells, effective to inhibit the infection of Tcells by the HTLV I virus. In one embodiment of this invention, theanimal is a mammal, e.g., a mouse or a human. Preferably, the mammal isa human.

It is well known to those skilled in the art that the CD4 protein is thecellular protein to which the HTLV I virus binds. HTLV I virus thusspecfically infects CD4⁺, but not CD8+, T cells. This invention providesa protein, the protein to which monoclonal antibody 5c8 binds, alsospecific to CD4⁺ T cells.

This invention provides a method of screening a pharmaceutical compound,e.g., cyclosporin, cyclophosphamide or azothioprine, for its ability toinhibit T cell helper function which comprises: isolating a sample ofblood from an animal; culturing said sample under conditions permittingactivation of the B cells cotained therein; contacting the sample withan amount of the D1.1 cell line effective to activate B cells;contacting the sample with an amount of a pharmaceutical compoundeffective to inhibit T cell activation of B cells if the pharmaceuticalcompund is capable of inhibiting T cell activation; and determiningwhether the T cell line activates B cells in the presence of thepharmaceutical compound.

In one embodiment of this invention, the B cells are resting B cells. Inanother embodiment of this invention, the B cells are primed B cells.

In one embodiment of this invention, the blood is isolated from amammal, e.g., a mouse or a human.

“Isolating” blood from an animal means any of the generally acceptablemethods of withdrawing blood and immediately placing the blood into areceptacle containing an anticoagulant, e.g., heparin, EDTA or citrate.Culturing B cells under “conditions permitting activation of B cells”comprises culturing B cells in the presence of lymphokines. An“effective activating amount” of the D1.1 cell line is any concentrationof the cells in culture effective to activate B cells in the culture.Methods of determining an “effective activating amount” are well knownto those skilled in the art.

A method of immunizing an animal against a protein antigen whichcomprises: isolating a sample of blood including immature B lymphocytesfrom the animal; recovering immature B cells from said sample;coculturing said immature B cells with an amount of the cell line D1.1or the pharmaceutical composition comprising the soluble activated Tcell surface protein effective to stimulate the B cells to differentiateunder conditions permitting the differentiation of B cells; contactingsaid differentiated B cells with an amount of the protein antigeneffective to induce the differentiated B cells to produce an antibodywhich recognizes the protein antigen; and administering saidantibody-producing B lymphocytes to the animal from which the bloodsample was isolated.

For the purposes of this invention, “immature B cells” areundifferentiated, non-antibody synthesizing B cells.

“Isolating” blood from an animal means any of the generally acceptablemethods of withdrawing blood and immediately placing the blood into areceptacle containing an anticoagulant, e.g., heparin, EDTA or citrate.Culturing B cells under “conditions permitting differentiation of Bcells” comprises culturing B cells in the presence of lymphokines.Methods of administering the B lymphocytes to the animal include any ofthe generally acceptable methods for administering cells to an animal.

An “effective amount” of the D1.1 cell line or the soluble activated Tcell surface protein is any amount of the cell line or the solubleprotein effective to induce B cells to differentiate. Methods ofdetermining an “effective amount are well known to those skilled in theart.

An “effective differentiating amount” of a protein antigen is any amountof the antigen effective to induce differentiated B cells to produce anantibody which specifically recognizes the antigen.

In one embodiment of this invention, the animal is a mammal, e.g., amouse or a human. Preferably, the mammal is a human.

In one embodiment of the invention, the antigen is a viral proteinantigen, e.g., a hepatitis B virus protein antigen, a Human T cellLeukemia Virus protein antigen or a Humnan Immunodeficiency Virusprotein antigen. In another embodiment of this invention, the antigen isan autoantigen.

This invention provides a method of treating a patient suffering fromhypogammoglobulinemia which comprises administering to the patient anamount of the soluble activated T cell surface protein effective totreat the patient for hypogammoglobulinemia. Methods of determining an“effective amount” are known to those skilled in the art.

Materials and Methods

GENERATION AND CHARACTERIZATION OF 5C8 MONOCLONAL ANTIBODY. Five Balb/cmice were immunized with 2×10⁶ D1.1 cells in saline intravenously andthen boosted intraperitoneally at five, approximately two-week,intervals. The sera of these mice were titrated to test for the presenceof antibodies that bound preferentially to Jurkat D1.1 versus B2.7 cellsby FACS. One mouse, which showed the best differential titer, received aboost of 2×10⁶ D1.1 cells intravenously 3 d prior to fusion. Splenocytesfrom this mouse were fused with 7×10⁷ murine SP2/0 myeloma fusionpartner cells as previously described (60). The cell mixture wascultured overnight in Dulbecco's Modified Eagle's Medium (DMEM)containing 15% FCS before the fusion product was seeded into 360 8-mmwells. Colonies appeared in 220 wells and all were screened by FACS fordifferential binding to D1.1 and B2.7 cells. A mAb designated 5c8 wasfound to bind to D1.1 cells and not B2.7 cells. The 5c8 clone wassubcloned multiple times until monoclonality was established. The 5c8mAb was found to be IgG2a by Elisa (Hyclone, Logan, Utah).

MONOCLONAL ANTIBODIES. The following mAbs were produced by hybridomasavailable from the American Type Culture Collection (Rockville, Md.):OKT11 (anti-CD2), OKT10(anti-38), OKT8(anti-CD8), OKT6(anti-CD1a),OKT4(anti-CD4), OKT3(anti-CD3), OKT1(anti-CD5), 3A1(anti-CD7),tac(anti-CD25), T-HB5(anti-CD21, CR2), W6/3Z(anti-MHC class I),AB2.06(anti-MHC class II), L243(anti MHC class II), 93F10(anti-MHC classII), TS1/22.1.13(anti-LFA-1a), TS1/18.1.2.11.4(anti-LFA-1β),TS2/9.1.4.3(anti-LFA-3) and 187.1(anti-human Ig(Fab)). These mAbs wereeither used at saturating concentrations of hybridoma supernatants, orpurified from ascites fluid on protein A columns (Biorad, RockvilleCenter, N.Y.). The anti-Jurkat TCR clonotypic (anti-v38) mAb 16G8 and apanel of other such anti-TCR mAb were purchased from Diversi-T, T CellScience (Cambridge, Mass.). The mAb OKT4A was purchased from OrthoPharmaceutical (Raritan, N.J.), TCR6-1 was the gift of Dr. MichaelBrenner, Harvard Medical School (Boston, Mass.). M241(anti-CD1c) was thegift of Dr. Cox Terhorst of Harvard Medical College.FITC labeledant-CD23-PE mAbs and unlabelled anti-CD69 were purchased from BectonDickinson (Mountainview, Calif.). FITC labeled anti-IgM was purchasedfrom Tago (Burlingame, Calif.).

Kolt-4 (anti-CD28) and anti-CD27 were purchased from Accurate Scientific(Westbury, N.Y.).

Recombinant proteins, rIL-4 was purchased from Genzyme (Cambridge,Mass.). rIL-2 was a gift of Hoffmann-LaRoche (Nutley, N.J.).

CYTOFLUOROGRAPHIC ANALYSIS. Approximately 10⁵ cells were incubated withsaturating concentrations of the indicated mAbs for 45 min at 4° C. inthe presence of 80 μg/ml heat-aggregated human IgG (InternationalEnzyme, Fallbrook, Calif.). Cells were washed to remove unbound mAbbefore incubation with goat anti-mouse Ig secondary antibody coupled tofluorescein (Cappel, Cochranville, Pa.).

For two color analysis, cells were reacted with the indicated directlycoupled FITC or Phycoerythrin (PE) conjugated mAb for 45 min at 4° C. inthe presence of aggregated human IgG. Prior to analysis, cells werewashed and resuspended in PBS. Fluorescence intensity was measured on aFACSCAN Cytofluorograph with the consort-30 software (Becton-Dickinson,Mountainview, Calif.). In experiments involving co-culture of B cellswith Jurkat clones, the Jurkat cells were excluded from the analysis ofB cell fluorescence by gating on the distinct population of cells withlow forward and side light scatter. In experiments with PMA and PHAactivated cells, dead cells were excluded from analysis by treatmentwith propridium iodide and electronic FACS gating.

CELL LINES. The following cell lines are available from the AmericanType Culture Collection (Rockville, Md.): HPB-ALL, Jurkat, CEM, PEER,MOLT-IV, K562, Ramos, Raji and U937. BA is an Epstein Barr virustransformed B cell line that has been previously reported (61). H9 isavailable from the HIV Repository (Rockville, Md.). HLA typings wasperformed by Dr. Elaine Reed of the Department of Pathology, ColumbiaUniversity (One Lambda, Los Angeles, Calif.). Jurkat D1.1 and B2.7 werenegative for mycoplasma by the Mycotect kit (GIBCO, Grand Island, N.Y.)and by the DNA hybridization method (Genprobe, La Jolla, Calif.).

ISOLATION OF CELL POPULATIONS. Peripheral blood lymphocytes wereobtained from the freshly drawn blood of healthy volunteers bycentrifugation on Ficoll-Hypaque (Sigma, St. Louis, Mo.) or Leukoprep(Becton-Dickson). T cells were positively selected with neuramimidasetreated sheep erythrocytes. CD4⁺CD8⁻ and CD4⁻CD8⁺ T cell subsets wereisolated by anti-CD8 or anti-CD4 mAb treatment, respectively, followedby complement mediated lysis as previously described (19). B cells werederived from the population of cells that did not pellet throughficoll-hypaque after two rounds of rosetting with neuramimidase treatedsheep erythrocytes.

B cells were further purified by either density centrifugation or bypositive selection on an anti-Ig column. In the first method, E− cellswere cultured overnight in polystyrene flasks (37° C., 5% CO₂) toadherence deplete macrophages. These non-T cell, non-macrophage cellswere fractionated into high and low density fractions in a discontinuous30%/50%/100% percoll gradient by centrifugation at 2300 rpm for 12 min.High-low-density cells were obtained from the 50/100% interface andlow-density cells from the 30/50% interface (62). The high density(resting) cells were typically 60-80% CD20⁺, 55-80% IgM⁺ and <5% CD3⁺and <5% CD23⁺ (background). In other experiments (where indicated) Bcells were purified by sephadex G-200 anti-F(ab)₂ Ig affinitychromatography into sIg⁺ cells as has been described (19, 63). ThesIg⁺populations were typically <5% CD3+, <10 CD2⁺ and >90% CD20⁺ whenanalyzed by FACS.

SDS POLYACRYLAMIDE GEL ELECTROPHORESIS. Jurkat clones were iodinated bythe lactoperoxidase method, solubilized in 1% NP40, 25 mM Tris BufferedPBS containing iodoacetamide and 10 μm PMSF. The cell lysates werereacted with protein A-4B Sephraose beads (Pharmacia, Uppsula, Sweden)that were coated with mAb 187.1 (anti-human F(ab)Ig) and approximately10 μg of the indicated mAb. After washing the beads to removenon-specifically bound proteins, the precipitated proteins weredenatured by heating in SDS in the presence or absence of 2-ME. Thedenatured proteins and pre-stained MW markers (Biorad, Rockville Center,N.Y.) were electrophoresed through 12% polyacrylamide in 12 cm gels(Biorad Protean Gel, Rockville Center, N.Y.) and dried gels were used toexpose X-ray film (Kodak, Rochester, N.Y.).

MITOMYCIN-C AND PARAFORMALDEHYDE TREATMENTS. Jurkat cells (10⁷/ml) weretreated with 50 μg/ml mitomycin-C (Sigma, St. Louis, Mo.) for 60′ min at37° C. The mitomycin-treated Jurkat cells were washed twice, resuspendedin mitomycin free media and then cultured for 45-60 min at 3° C. Thecells were washed two additional times and then added to the B cellcultures. In fixation experiments, T cells were treated with freshlymade 0.5% paraformaldehyde for 5-10 minutes, quenched with 0.2 ML-lysine and washed five times before addition to cultures of B cells.

T CELL ACTIVATION. In experiments studying expression of 5c8 Ag, restingT cells were cultured in the presence or absence of 10 μg/ml phorbolmyristate acetate (PMA) (Sigma, St. Louis, Mo.) and 10 ug/ml PHA(Sigma). In experiments studying the metabolic requirements for 5c8 Agexpression, T cells were activated in the presence of 100 μmcyclohexamide (Sigma) or 10 μg/ml actinomycin D (Sigma).

In experiments studying the induction of CD23 expression on high densityB cells by activated T cells, the mabs OKTs or OKT4 were immobilized onthe surfaces of 24 well culture plates by incubation of 10 μg/ml of mAbin PBS for 1 h. Control wells were incubated in PBS containing no mAb.After washing unbound mAb coated plates at 2×10⁶ cell/well in thepresence of 10 ng/ml phorbol dibutyrate (PDB) (Sigma) for 6 h. The cellswere removed by vigorous pipetting, washed and fixed with 0.5%paraformeldehyde as described above before culture at a ˜1:1 ratio with2×10⁵ high density, percoll isolated, resting B cells for 18 h. B cellCD23 expression was determined by 2-color FACS as described above.

ASSAYS OF B CELL ACTIVATION AND DIFFERENTIATION. In experimentsmeasuring the induction of B cell surface CD23 expression, 2×10⁵ highdensity B cells were added to the indicated number of Jurkat cells or Tcells in 200 μl of Isocove's Modified Dulbecco Medium (IMDM) 10% FCSround bottom microtiter wells (Nunc) and assayed for CD23 expressionafter 18-24 h. Two chamber experiments were performed with 5×10?? Jurkatcells in the presence or absence of 5×10?? B cells separated from 5×10⁵cells by 45-μm culture plate inserts from Millipore (Bedford, Mass.).

B cell proliferation was measured by culturing 10⁵ B cells with equalnumbers of mitomycin-C-treated E⁺ cells or Jurkat clones in flat bottommicrotiter wells (NUNC) in the presence or absence of PHA (5 μg/ml). Thecultures were pulsed with 1 μCI (H³) thymidine (New England Nuclear,Boston, Mass.) after 60 h and harvested 16 h later on glass fiber filterpaper (Cambridge Technology, Watertown, Mass.). Beta scintillation cpmwere measured on an beta counter (LKB Rackbeta counter, Model 1209).

The measurement of plaque forming colonies (PFC) was a modification ofthe Jerne hemolytic plaque assay (19). Briefly, 2.5×10⁵ B cells werecultured with varying numbers of mitomycin-C treated Jurkat cells oruntreated freshly isolated, autologous T cells for 6 days in thepresence or absence of a 1:400 dilution of PWM (Gibco, Grand Island,N.Y.). The cells were washed twice and resuspended in Hanks balancedsalt solution. From an appropriate dilution, 50 ul of cultured cellsuspension was mixed with: 10 μl of an 11% solution of SRBC that hadbeen coated with rabbit anti-human Ig by chromic chloride, 10 μl ofdiluted rabbit anti-human Ig and 10 μl of guinea pig complement. Thesemixtures were introduced into duplicate glass chambers and cultured for2 h at 37° C. Plaques were counted using a dissecting microscope andexpressed as plaque forming colonies (PFC) 10⁶ B cells.

ELISA for Ig isotype quantitation were performed by coating polystyrene96-well plates (immunion II, Dynatech Laboratories, Chantilly, Va.) withdilutions of goat anti-human IgA, IgG, or IgM (Tago, Burlingame, Calif.)in carbonate buffer, pH 9.6, for 18 h at 4° C. The plates were washedwith 0.05% Tween in PBS, and nonspecific sites were blocked by a 2 hincubation of 1% BSA-PBS. After washing, 50 μl of cell culturesupernatants or Ig isotype standards (Rockland, Gilbertsville, Pa.) wereadded to the wells and allowed to bind for 2 h. Next, goat anti-human Igcoupled to alkaline phosphatase (Tago) was added to detect bound humanIg. After 2 h, the wells were washed and p-nitrophenyl phosphate wasadded. Absorbance was measured at 405 nm in a Molecular Devices VMAXdevice (Palo Alto, Calif.). Samples were assayed in triplicate. Errorbars represent calculated standard deviation from curve fit andinterpolation (Delta-Soft, BioMetallics, Inc. Princeton, N.J.).

EXAMPLES Example 1

Role of CD4 in T Cell Function

To study the role of CD4 in T cell functions, a CD4⁻ Jurkat clone (D1.1)was isolated from a culture that spontaneously developed a CD4⁻subpopulation identified by a negative peak on FACS analysis. The lackof CD4 surface expression was relatively specific in that the cellsurface phenotype of Jurkat D1.1 with respect to the binding of a largepanel of mAb was similar to a CD4⁺ clone, Jurkat B2.7 (FIG. 1 and Table1). Although the differential expression of CD4 was the only qualitativedifference between these subclones, some of the other molecularstructures studied were expressed at quantitatively different levels.For example, Jurkat D1.1 expressed more CD2 and MHC class (HLA)molecules than Jurkat B2.7. However, Jurkat D1.1 expressed fewer CD28molecules and fewer TCR-α/β(vβ8)/CD3 complexes than Jurkat B2.7 (FIG. 1and Table 1). In addition to their shared reactivity with the clonotypicanti-TCR mAb, Jurkat D1.1 and B2.7 were HLA identical (A3, 34, 2, 16)and distinct from an unrelated T cell leukemic line, HPB-ALL (A9).Together, these data demonstrated that Jurkat D1.1 was a CD4⁻ subcloneof Jurkat and that the absence of CD4 molecules was a relativelyspecific alteration in its surface phenotype. TABLE 1 CELL SURFACEPHENOTYPES OF JURKAT CLONES D1.1 AND B2.7 Mean FluorescenceIntensity^(a) CD No. Molecule mAb D1.1 B2.7 TCRα/β BMA-031 10 40 TCRvβ816G8 30 70 TCR-vβ5 W6/32 0 0 MHC-classI W6/32 190 70 MHC-classII 2.06 00 CD1a T6 OKT6 10 10 CD1c M241 10 10 CD2 T11 OKT11 100 10 CD3 TCRcomplex OKT3 30 80 CD4 T4 OKT4 0 130 CD5 T1 OKT1 20 90 CD7 3A1 200 190CD8 T8 OKT8 0 0 CD11a LFA-1α TS1/22.1.13 40 100 CD14 My2 0 0 CD16 FcyRII3G8 20 20 CD18 LFA-1β TS1/18.1.2.11.4 30 80 CD21 CR2 HB-5 0 0 CD23FcyRII leu20 0 0 CD25 tac. IL-2Rα tac 0 0 CD26 DPPIV taq-1 0 0 CD28 9.3.gp44 KOLT-4 30 70 CD29 4B4 140 110 CD38 T10 OKT10 40 30 CDw32 FcyRII32.2 0 0 CD45RA T200. LCA 2H4 30 40 CD45RO T200. LCA UCHL1 10 20 CDw49VLA-1 1B.3 0 0 CD58 LFA-III TS2/9.1.4.3 40 60 CD64 FcyRI IV 3 0 0^(a)Numbers represent mean fluorescence intensity (arbitrary units) asdetermined by FACS. Background is subtracted and numbers are rounded offto the nearest ten units.

In functional studies, we compared the ability of CD4⁺ (B2.7) and CD4⁻(D1.1) Jurkat cells to induce resting B cells to express CD23, a markerof B cell activation (32, 35, 57). Surprisingly, co-culture of B cellswith CD4⁻ Jurkat (D1.1) but not CD4⁺ Jurkat cells (B2.7) induced CD23expression on greater than 60% of B cells (FIG. 2). The induction of Bcell surface CD23 expression by Jurkat D1.1 was maximal at 20-24 h at aratio of 1:1 D1.1 cells to B cells (FIG. 3). In contrast, the B3.7Jurkat subclone did not activate B cells at high ratios (FIG. 3) or atlong periods of coculture (up to 48 h, not shown). In addition, JurkatD1.1 was unique in this ability compared with other T cell (H9, HPB-ALL,MOLT-IV, CEM) and non-T cell (U937) leukemic lines (not shown). JurkatD1.1 induced B cell CD23 expression selectively because the levels ofother B cell surface molecules such as IgM. (FIG. 2), CD20 (FIG. 2), orclass I MHC were not affected. The effect of Jurkat D1.1 on B cellactivation was consistently observed on B cells from over 25 unrelateddonors, suggesting that the effect was neither Ag nor MHC restricted.

B cell CD23 expression is an early and possibly intermediate stage interminal B cell differentiation into Ig-secreting cells. Other stimuli,besides those contributed by activated T cell surfaces are required tomediate substantial B cell proliferation and differentiation. Becausethe measurements of B cell proliferation or differentiation requireseveral days of culture, we inhibited the proliferation of the Jurkatclones by pretreatment with mitomycin-C, which did not abolish theircapacity to activate B cells (Table 2). TABLE 2 EFFECTS OF MITOMYCIN-CAND ANTIBODIES TO IL-4 ON B CELL CD23 EXPRESSION INDUCED BY JURKAT D1.1CELLS B cells Jurkat clones plus C rIL-4 rIL-2 D1.1 B2.7 D1.1/M B2.7/M14 64 17 81 16 57 14 Anti-IL-4 ND 28 ND 84 ND 64 ND Anti-IL-2 ND 60 ND86 ND 60 NDShown are the percentages of CD20(Leu-16)⁻ B cells expressing CD23 asdetermined by two-color FACS analysis with anti-CD20(Leu-16)-FITC andanti-CD23 PE. High density Percoll-fractionated B cells (2 × 10⁴) werecultured alone or with an equal number of either Jurkat B2.7 or D1.1cells as indicated for 20 h. Where indicated, purified polyclonal rabbitanti-IL-4 or anti-IL-2 Ig was added at the initiation# of the experiment to final concentrations of 1.25 μg/ml. Whereindicated, rIL-2 or rIL-4 were added to indicated cultures to finalconcentrations of 25 U/ml. Cells analyzed were gated by forward and sidelight scatter to exclude the larger D1.1 or B2.7 cells (when present)from the analysis.C: Control;D1.1/M: D1.1 cells treated with mitomycin-C;B2.7/M: B2.7 cells treated with mitomycin-C;ND: not determined.

Mitomycin-C treated CD4⁻ Jurkat D1.1 and CD4⁺ Jurkat B2.7 were thenstudied for their ability to induce B cell proliferation or terminal Bcell differentiation into Ig-secreting cells. In the presence of Tcell-dependent B cell mitogens (66), Jurkat D1.1- but not B2.7-induced Bcell proliferation measured by DNA synthesis (FIG. 4) anddifferentiation to Ig-secreting cells measured by reverse hemolyticplaque assay (FIG. 5A). In addition, the isotype of secreted antibodywas characterized by quantitative ELISA. Jurkat D1.1 but not B2.7induced the secretion of IgG and to a lesser extent, IgM into theculture supernatant (FIGS. 5B and C). Taken together, these data showthat Jurkat D1.1 but not Jurkat B2.7 shared with activated T cells thefunctional capacity to support B cell differentiation and the secretionof IgM and IgG.

Example 2

Role of Diffusible Factors in B Cell Activation

D1.1 supernatants did not induce B cell CD23 expression (FIG. 3). Wenext performed two chamber experiments in which resting B cells werecultured in a chamber that was separated by a permeable membrane fromeither lymphokine containing media or from cultures of D1.1 cells in thepresence or absence of B cells. In an experiment in which B cells (66%IgM⁺) were cultured in a chamber with a 0.45-mμ membrane. rIL-4 (25U/ml) induced CD23 expression on 28% of IgM⁺ B cells by two-color FACSanalysis. In contrast, D1.1 cells did not activate B cells in the otherchamber to express CD23 (4.7% for D1.1 vs 4.0% background). In addition,coculture of D1.1 cells with B cells in one chamber did not activate Bcells in the other chamber to express Cd23 (4.9%). However, D1.1 cellspotently induced CD23 expression by the B cells with which they couldestablish direct contact (76% vs 8.4% for B2.7 cells). Taken together,these data failed to support a role for diffusible factors in mediatingthe D1.1 effect on B cells.

Because rIL-4 was known to activate B cells to express CD23 (67), wefurther studied the potential role of IL-4 in mediating this effect inaddition to inducing CD23 expression on B cells. rIL was known toup-regulate B cell sIgM⁺ expression (59). Whereas rIL-4 induced CD23expression and sIgM up-regulation in a dose-dependent manner, D1.1 cellsinduced CD23 expression but did not up-regulate B cell sIgM (FIG. 6).The effect of D1.1 cells on B cell proliferation was also distinct fromthat of rIL-4 (FIG. 4). D1.1 cells, but not rIL-4 induced B cellproliferation in the presence of PHA. Interestingly, rIL-4 and D1.1cells collaborated to induce B cell proliferation in the absence of PHAand augment D1.1 induced proliferation in the presence of PHA. Takentogether these data suggest that the effect of D1.1 cells on B cells aredistinct from those induced by I-4. However, to directly examine therole of Il-4 in D1.1's effect on B cells, we used neutralizingantibodies to Il-4. Concentrations of anti-IL-4 antibodies thatinhibited both the CD23 induction and sIgM up-regulation mediated byrIL-4 (FIG. 6) did not inhibit D1.1-mediated B cell CD23 expression(Table II). These data demonstrated that IL-4 alone did not account forthe effect of D1.1 on B cells. Taken together, these results stronglysuggested that cell-cell contact and not secreted factors accounted forthe effects of D1.1 on B cell activation.

To substantiate the idea that cell-cell contact mediated the D1.1 effecton B cells, we fixed Jurkat D1.1 and control, B2.7 cells with 1%paraformaldehyde. Although paraformaldehyde fixation decreased thepotency of Jurkat D1.1 to activate B cells, fixed D1.1 cells remainedcompetent to induce B cell CD23 expression whereas, fixed B2.7 cells didnot alter CD23 expression from the background level. At a ratio of 5:1fixed D1.1 cells:B cells, 63% of B cells were induced to express CD23 ascompared with 80% for unfixed D1.1 cells. Taken together, these datasuggest that that surface structures on Jurkat D1.1 are sufficient toinduce B cell activation.

Example 3

Characterization of Cell Surface Proteins on Activated CD4⁺ T Cells thatMediate Helper Effector Function

In order to characterize cell surface proteins on activated CD4⁺ T cellsthat mediate helper effector function, mice are immunized with the D1.1clone of Jurkat that possess contact dependent helper effector function(58). Monoclonal antibodies (mAb) are generated and hybridomasupernatants are screened for differential binding to the D1.1 clone anda non-helper Jurkat clone, B2.7.

A murine IgG2a mAb, termed 5c8, was identified that bound specificallyto the surface of D1.1 cells and not to the surface of the non-helper,B2.7 cells (FIG. 7). The mAb 5c8 did not bind to a variety of other celllines including: the T cell leukemia lines, CEM, H9, Molt-4 and Peer;the B cell derived cell lines, BA, Raji or Ramos; the myelomonocyticcell line, U937; or the erythroleukemia cell line, K562 (see Table 3below). TABLE 3 EXPRESSION OF 5c8 Ag ON CELL POPULATIONS AND CELL LINESResting Activated Cell Lines Jurkat D1.1 + + Jurkat B2.7 − − CEM − − H9− ND Molt-4 − − PEER − − BA − ND Raji − ND Ramos − ND U937 − − K562 − NDCell Populations T cells − + B cells − − Monocytes − −These data derive from FACS analyses of mAb 5c8 binding to the indicatedcell lines or cell populations. The presence of mAb 5c8 binding wasdetermined relative to FACS staining of appropriate positive andnegative control mAbs for each cell line or population.Nd: Not determined.

To assess whether mAb 5c8 reacts with a molecule that is functionallyrelevant to the helper capacity of the Jurkat clone D1.1, the effect ofmAb 5c8 is studied in assays of D1.1 induced CD23 expression on B cells.The mAb 5c8 potently inhibited Jurkat D1.1 induced cell activation (FIG.8). In contrast, the isotype control mAb, W6/32 did not inhibit D1.1mediated B cell activation. The data presented here suggest that the 5c8Ag plays a critical role in the helper effector function of D1.1 cells.

Example 4

Biochemical Characterization of the Antigen Recognized by mAb 5c8

In order to biochemically characterize the antigen recognized by mAb5c8, immunoprecipitations are performed with mAb 5c8 or control mAbsthat recognized Class I MHC (W6/32) or CD28 (Kolt-4) antigens on celllysates of surface iodinated Jurkat D1.1 cells and control, non-helperJurkat B2.7 cells that lack surface mAb 5c8 binding. The mAb 5c8immunoprecipitated a protein that migrated on SDS/PAGE at 30 kDa fromlysates of the helper clone D1.1 but not from the control B2.7 lysates(FIG. 9).

The protein species immunoprecipitated by mAb 5c8 was not affected byreduction with 2-mercaptoethanol (2-ME) suggesting that the 30 kDa bandwas neither a disulfide linked homodimer nor disulfide linked to anotherprotein that was not accessible to iodination. In contrast, the control,anti-CD28 mAb, KOLT-4 immunoprecipitated (FIG. 9) an 88 kDa band in theabsence of 2-ME and a 44 kDa band in the presence of 2-ME that isconsistent with published reports (64) and with the interpretation thatthis structure is a disulfide linked homodimer. The control mAb W6/32precipitated a non-disulfide linked heterodimer of 43 and 12 kDa MWproteins (FIG. 9). These data suggested that the mAb 5c8 recognized a 30kDa MW non-disulfide linked protein species from the surface of D1.1cells.

Example 5

Characterization of the Expression of 5c8 Ag by Normal Lymphoid Cells

The binding of mAb 5c8 or a variety of control mAbs is studied by FACSon freshly isolated, T and B lymphocytes, monocytes and PMA and PHAstimulated T cells. Although, resting T or B lymphocytes or monocytesdid not express 5c8 Ag (see Table 3 above and FIG. 10), a subset ofactivated T cells was found to express 5c8 Ag, 5 h after activation withPMA and PHA (FIG. 10).

To characterize the kinetics and cellular distribution of 5c8 Agexpression, the binding of mAb 5c8 to T cells was studied by FACS atvarious intervals after T cell activation. The CD69 molecule, which is a32/28 KDa disulfide linked heterodimer, is selected as a control becauseit is known to be induced rapidly on virtually all T cells after T cellactivation (65). Whereas 5c8 was absent from resting T cells and wasexpressed on a subset of T cells following activation, in contrast, lowlevel CD69 expression was present on resting T cells and high level CD69expression was induced by activation on the entire T cell population(FIG. 10). The kinetics of expression further distinguished 5c8 Ag fromCD69 because mAb 5c8 binding was significant 3 h after activation (65)and persisted for over 24 h (FIG. 11). The data presented heredistinguish the 5c8 Ag from CD69 both by the cellular distribution oftheir expression and by the kinetics of their up-regulation followingactivation.

To determine if mRNA or protein synthesis is required for 5c8 Agexpression, T cells are stimulated by PMA and PHA in the presence orabsence of Actinomycin D or cyclohexamide and the expression of 5c8 andCD69 was compared. The expression of 5c8 was inhibited by eitheractinomycin D or cyclohexamide treatment (FIG. 10). In contrast, CD69was up-regulated by activation despite the presence of actinomycin D orcyclohexamide (FIG. 11), as has been reported previously (65). Thesedata suggested that the expression of the 5c8 antigen after T cellactivation depends on transcription of mRNA and de novo proteinsynthesis.

Example 6

Characterization of the Subset of T Cells that Express 5c8 Ag AfterActivation

In order to characterize the subset of T cells that expressed 5c8 Agafter activation, CD4⁺CD8⁻ or CD4⁻CD8⁺ T cell populations were isolatedby anti-CD8 or anti-CD4 mAb treatment, respectively, followed bycomplement depletion. The CD4⁺CD8⁻ or CD4⁻CD8⁺ populations wereactivated with PHA and PMA and studied for 5c8 Ag or CD69 expression byFACS. After activation, 5c8 expression was induced exclusively on CD4⁺cells and not on CD8⁺ cells, despite the fact that CD8⁺ cells expressedsimilar levels of CD69 after activation (FIG. 11). Taken together, thesedata demonstrated that 5c8 Ag expression is restricted to activated CD4⁺cells.

Example 7

Evaluation of the Role of 5c8 Ag in T Helper Function Mediated by NormalT Cells

To evaluate the role of 5c8 Ag in T helper function mediated by normal Tcells, the effect of mAb 5c8 was studied on the ability of activated Tcells to induce small resting B cells to express surface CD23 molecules.T cells were cultured on surfaces that were coated with anti-CD3(OKT3)or control, anti-CD4(OKT4) mAbs in the presence of phorbol dibutyrate(PBD) and then fixed with paraformaldehyde. These fixed T cells werestudied for B cell activating capacity in the presence of soluble mAb5c8 or OTK4. The mAb OKT4 was selected as an isotype matched control inthese experiments because OKT4 reacts with T cell surface CD4 moleculesbut does not inhibit T-B interactions (19). The mAb 5c8, but not OKT4inhibited the ability of activated T cells to induce B cell CD23expression (see Table 4 below). TABLE 4 EFFECT OF mAb 5C8 TREATMENT ON BCELL SURFACE CD23 INDUCTION MEDIATED BY PARAFORMALDEHYDE FIXED,ACTIVATED T CELLS. Media mAb 5c8 OKT4 No T cells 6.8 ND ND Jurkat D1.193.8  9.8 96.1 PDB-activated T cells 29.8 ND ND PDB/OKT4-activated Tcells 26.0 ND ND PDB/OKT3-activated T cells 52.7 30.4 56.1Shown are the percentages of IgM⁺ B cells that expressed CD23 by 2-colorFACS analysis after B cells were cultured alone or in the presence ofequal number of Jurkat D1.1 cells or paraformaldehyde fixed T cells thathad been stimulated with PBD alone or in the presence of eitherimmobilized anti-CD3 (OKT3) or anti-CD4 (OKT4) mAbs, as indicated. TheIgG2a mAbs, 5c8 and OKT4 were present at 500 ng/ml which is twice theconcentration of mAb 5c8 that# inhibited 90% of CD23 induction in a parallel dose responseexperiment.ND: Not determined.

The effect of mAb 5c8 was next compared to that of OKT4 for its abilityto inhibit terminal B cell differentiation driven by normal human Tcells. In these experiments, CD4⁺ T cells were cultured with autologous,column isolated B cells in the presence of PWM and the number of Igsecreting B cell plaque forming colonies (PFCS) was measured by reversehemolytic plaque assay. The mAb 5c8, but not OKT4, inhibited the CD4⁺cell driven PFC response (see Table 5 below). Taken together, these datademonstrated that the 5c8 Ag mediates a contact dependent aspect of thehelper effector function of activated CD4⁺ T cells. TABLE 5 EFFECT OFmAb 5C8 TREATMENT ON THE INDUCTION OF ANTIBODY FORMING CELLS PFC T cellsB cells PWM mAb Exp. 1 Exp. 2 Exp. 3 B 120 240 600 B PWM 240 600 4,800CD4⁺ T 240 120 180 CD4⁺ T B 2,580 780 ND CD4⁺ T PWM 3,840 240 60 CD4⁺ TB PWM 149,760 85,200 25,800 CD4⁺ T B PWM 5c8 58,000 4,680 9,000 CD4⁺ T BPWM OKT4 143,520 103,200 30,960Shown are the results of three separate experiments on unrelated donorsin which CD4⁺ T cells were cultured in a 0.6:1 ratio with autologcus,anti-Ig column isolated B cells in the presence or absence of PWM. Thenumber of plaque forming colonies (PFC) per 106 B cells was measured byreverse hemolytic plaque assay. The mAbs 5C8 and OKT4 were present at500 ng/ml except in experiment 1., in which OKT4 was present at 1 ug/ml.ND: Not determined.Discussion

The Jurkat D1.1 clone is functionally distinct from CD4+ Jurkat and froma variety of other leukemic T cell lines in that it induced B cells fromvariety of unrelated subjects to express surface CD23 molecules, amarker of B cell activation and to proliferate and terminallydifferentiate into ISC in the presence of T-dependent B cell mitogens.The effect of D1.1 on B cell activation required intimate cellularcontact and could not be accounted for by secreted factors or by IL-4 inparticular. The fact that Jurkat D1.1 was able to induce contactdependent B cell activation and differentiation suggested that JurkatD1.1 shares surface structure(s) with activated T cells that mediate thecontact-dependent, effector phase of help.

The molecular interactions between activated T cells and B cells thatmediate the effector phase of T helper function is complex and poorlyunderstood. To dissect the mechanism of T helper effector function,several studies have measured early events in B cell differentiation.First, B cell synthesis of RNA, DNA and enzymes associated with cellcycle progression are induced by activated but not resting T cells (6,8, 9, 11, 13-18). Second, B cell activation, measured by the inductionof B cell surface CD23, is induced by activated but not resting T cells(13). Third, B cell activation and proliferation can be induced byactivated T cells that have been fixed with paraformaldehyde (13, 17).Fourth, B cell proliferation is induced by membrane preparations fromactivated but not resting T cells (9, 12, 33). Finally, the ability ofactivated T cells or activated T cell membranes to induce B cellactivation or proliferation is abrogated by protease treatment (12, 16).Taken together, these observations are consistent with the idea that Tcell activation is associated with the induction of a surface structurethat interacts with B cells and provides a contact dependent signal forB cell activation and proliferation. Similar to activated T cells, butunlike other leukemic cell lines, Jurkat D1.1 had the capacity to induceB cell CD23 expression in a manner that depended on cell-cell contactbut was independent of lymphokines, Ag specificity or MHC restriction.The induction of B cell surface CD23 expression appears to be an earlyor intermediate stage in T-directed B cell differentiation into Igsecreting cells that can be driven by the surfaces of fixed, activated Tcells (13, 14). In addition to inducing B cell CD23 expression, JurkatD1.1 was functionally distinct from CD4+ Jurkat clones in that D1.1induced terminal B cell differentiation in the presence of PWM. In theserespects, Jurkat D1.1 appears to have acquired surface features that itshares with activated T cells and that stimulate B cells.

The nature of the structure on Jurkat D1.1 that accounts for helperfunction was not identified in the present work. Because CD28 moleculeson T cells bind a B cell ligand (34), it was of particular interest tocompare the expression of. CD28 on the helper D1.1 and non-helper B2.7clones. However, the fact that both Jurkat D1.1 and B2.7 expressed CD28molecules demonstrated that CD28, alone, could not account for theunique functional properties of Jurkat D1.1. Moreover, in antibodyblocking studies using mAb specific for CD2, CD3, CD5, CD38, LFA-1a,LFA-1b and LFA-3; we were unable to identify mabs that inhibited D1.1mediated B cell activation (not shown). In order to identify thedistinctive cell surface features of D1.1 that mediate helper effectorfunction, we have initiated an attempt to generate mAbs that react withD1.1 and inhibit D1.1 's ability to help B cells.

Although the surface structures that mediate helper function were notidentified, the D1.1 system is instructive with respect to the role ofCD4 molecules in helper effector function. It is curious that a Jurkatsubclone isolated for being CD4⁻ possessed helper function, which isnormally associated with the subset of T cells that express CD4molecules (14, 35). Several lines of investigation have suggested thatCD4 molecules do not play a direct role in helper effector function(2,8-12). However, the fact that both TCR and CD4 are known to interactwith MHC Class II molecules (Ia) (36) have suggested that ligation of Iamolecules might be a model for helper effector function. In addition,the observation that ligation of Ia molecules on B cells can signal Bcells has further supported this model (37-39). The fact that JurkatD1.1 had helper function but was CD4− strongly suggests that CD4molecules are not required for the effector phase of helper function. Onthe contrary, the finding that a CD4− clone of Jurkat has acquiredhelper function suggests that CD4 molecules might inhibit the helpereffector function of CD4+ Jurkat cells. In order to directly determinethe relationship between the lack of CD4 molecules on Jurkat D1.1 andits unique helper function, we have generated stable CD4+ transfectantsof D1.1 by electroporation of CD4 cDNA constructs driven by heterologouspromoters. The expression of CD4 did not inhibit the ability of D1.1transfectants to activate B cells suggesting that D1.1 's helperactivity is mediated by surface features other than the lack of CD4molecules.

Recently it has been shown in the murine system that membranepreparations derived from activated, but not resting T lymphocytes aresufficient to induce B cell proliferation but not Ig secretion (9, 12,33). The relevance of these studies to the D1.1 system is presentlyunclear, but it will be of interest to determine if membranes isolatedfrom D1.1 cells induce B cell CD23 expression., proliferation andterminal differentiation. In any case, it is likely that Jurkat D1.1will be useful for the identification and characterization of surfacemolecules important in mediating contact dependent helper function.

A functionally unique Jurkat leukemic line (D1.1) with constitutive,contact dependent helper function was utilized to generate a murine mAb,designated 5c8, that inhibited D1.1 induced B cell activation. The mAb5c8 recognized a unique protein species on D1.1 cells that was notdisulfide linked and migrated at 30 kDa MW on SDS/PAGE. On normallymphoid cells, the expression of 5c8 Ag was restricted to a subset of Tlymphocytes after activation. The activation induced expression of 5c8Ag on T cells required transcription of mRNA and de novo proteinsynthesis. The 5c8 Ag was found to be transiently expressed on activatedT cells with peak expression at 6 h and loss of expression by 24 h. Theexpression of 5c8 Ag was restricted exclusively to activated CD4+ Tcells. In functional studies on normal T cells, the mAb 5c8 inhibitedthe ability of fixed, activated T cells to induce B cell CD23expression. In addition, mAb 5c8 inhibited the ability of normal T cellsto direct B cell differentiation. Taken together, these data demonstratethat the 5c8 Ag is a novel activation-induced surface protein expressedexclusively on activated CD4⁺ T cells that is involved in mediating acontact dependent element of T helper function.

The tissue distribution, kinetics of expression, metabolic requirementsfor induction and biochemistry of the 5c8 Ag distinquished the 5c8 Agfrom other known surface proteins induced by T cell activation. First,all other known T cell activation markers (e.g. CD69, CD25, Ia) areexpressed by both CD4+ and CD8+ T cells whereas the 5c8 Ag is expressedexclusively by CD4⁺ T cells. Second, the kinetics of 5c8 Ag expressionfollowing T cell activation were distinct from that of other T cellactivation molecules. Whereas 5c8 Ag was maximally expressed 6 h afteractivation and absent 24 h after activation, CD25 (66), Ia (67, 68) andthe 32 kD form of CD27 (69) are induced 18 h or more after activation.In addition, CD69 is expressed more rapidly than 5c8 Ag and (unlike 5c8Ag) persists for over 24 h. Third, 5c8 Ag was distinquished from CD69 bythe metabolic requirements of their induction, because induction of 5c8Ag but not CD69 expression depended on mRNA transcription and proteinsynthesis. Fourth, the 5c8 Ag was a 30 kD, non-disulfide linked species.In contrast, the early activation molecule, CD69 is a 28/32 kD disulfidelinked heterodimer (65). Taken together, these data suggest that the 5c8Ag was distinct from other known T cell activation molecules.

The 5c8 Ag was also distinquished from other T cell surface moleculesthat are known to play roles in T-B interactions by several aspects oftheir tissue distribution and biochemistry. First, 5c8 Ag was induced byT cell activation but was not expressed on resting cells. In contrast,CD4, CD2, CD5, CD28, LFA-1, ICAM-1, CD45RO and 6C2, which interact withB cell surface ligands (70-78) are expressed on resting T cells (77-82).Second, the specific expression of 5c8 Ag on activated T lymphocytes andnot on B cells, monocytes or the panel of cell lines (Table 1.)distinguished 5c8 Ag from ICAM-1, CD4, CD5, LFA-1, CD2 and 6C2 moleculeswhich are also expressed on either monocytes, B cells or certain of thecell lines (not shown). Third, the expression of 5c8 Ag was restrictedto CD4⁺ T cells whereas CD2, CD5, CD28, LFA-1, ICAM-1, CD45RO and 6C2are expressed on CD8+ as well as CD4+ cells (77-82). Fourth, the 30 kDprotein precipitated by mAb 5c8 is unlike any of these other proteins(77-82). Finally, 5c8 Ag was distinct from these other molecules becausethe mAb 5c8 was identified by its ability to inhibit the helper effectorfunction mediated by Jurkat D1.1.

Because the mAb 5c8 inhibits the contact dependent helper effects ofJurkat D1.1 and fixed, activated T lymphocytes, it is likely that the5c8 Ag mediates a B cell activating function by interacting with aligand (or “counter-receptor”) on the surfaces of B cells. Theinteraction of 5c8 Ag with a B cell counter receptor may mediate helperfunction either by providing additional adhesive forces to T-B pairs,transducing a stimulatory signal to B cell cytoplasms or by acombination of these mechanisms. Regardless of the precise mechanism,the transient expression of 5c8 Ag may provide a molecular solution tolimiting non-specific B cell activation. We envision that the transientexpression of 5c8 Ag in the localized milieu of antigen specific cognateT-B pairs may channel the antigen/MHC unrestricted activating functionof 5c8 Ag to appropriate B cell targets. The kinetics of expression anddown-regulation of 5c8 Ag are shared by the endothelial cell, activationinduced, cell surface mediator of leukocyte and lymphocyte binding,ELAM-1 (83). This similarity might indicate that the strategy ofutilizing transient expression to effect localized intercellularinteractions may be shared by 5c8 Ag, ELAM-1 and potentially other, yetuncharacterized, surface molecules that transmit potent signals to othercells by direct contact.

The CD4 molecule identifies the population of T cells that containsprecursors of T cells with helper function (4). However, the CD4+ subsetis functionally heterogeneous and contains cytotoxic and suppressorcells in addition to helper cells (84, 85). The fact that 5c8 Ag isinvolved in helper function suggests that 5c8 Ag may correlate moreclosely with the helper phenotype than CD4 expression. The heterogeneousdistribution of 5c8 expression on activated CD4+ cells suggests thatfunctional subsets of CD4+ T cells might be distinguished by their levelof 5c8 expression. For example, it will be of interest to determine thefunctional potential of 5c8− and 5c8+ CD4+ T cells with respect tohelper or cytotoxic activity.

T cell helper effector function is a complex process resulting in B cellresponsiveness (22, 54-56), regulation of isotype switching (86) andsomatic hypermutation (87). The fact that T cells interact with B cellsby a number of cell-cell interactions as well as by secreting variouslymphokines suggests that individual signals or certain combinations ofsignals may regulate specific aspects of B cell differentiation. Thefact that the mAb 5c8 inhibits a contact dependent aspect of T cellhelper function provides a means of further dissecting the processes bywhich CD4+ T cells regulate the humoral immune response.

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1. A monoclonal antibody capable of binding to a protein which isspecifically recognized by monoclonal antibody 5c8 produced by thehybridoma having ATCC Accession No. HB
 10916. 2. (Canceled)
 3. Themonoclonal antibody of claim 1, wherein the monoclonal antibody isselected from the group consisting of chimaeric monclonal antibodies,humanized monoclonal antibodies, and human monoclonal antibodies. 4-7.(Canceled)
 8. A hybridoma cell producing the monoclonal antibody ofclaim
 1. 9-11. (Canceled)
 12. The monoclonal antibody of claim 1conjugated to a therapeutic agent. 13-15. (Canceled)
 16. Apharmaceutical composition comprising the monoclonal antibody of claim 1and a pharmaceutically acceptable carrier.
 17. An isolated nucleic acidmolecule encoding the light chain protein of the monoclonal antibody ofclaim
 1. 18. (Canceled)
 19. An isolated nucleic acid molecule encodingthe heavy chain protein of the monoclonal antibody of claim
 1. 20-23.(Canceled)
 24. An isolated protein which is specifically recognized bymonoclonal antibody 5c8 produced by the hybridoma having ATCC AccessionNo. HB
 10916. 25-31. (Canceled)
 32. An isolated nucleic acid moleculeencoding a fragment of the isolated protein of claim
 24. 33-43.(Canceled)
 44. An isolated, soluble protein which is specificallyrecognized by monoclonal antibody 5c8 produced by the hybridoma havingATCC Accession No. HB
 10916. 45-49. (Canceled)
 50. A pharmaceuticalcomposition comprising the soluble activated T cell surface protein ofclaim 44 and a pharmaceutically acceptable carrier. 51-58. (Canceled)59. A method of inhibiting the activation of B cells in an animal whichcomprises administering to an animal an effective amount of thepharmaceutical composition of claim 16 or 50 to inhibit the activationof B cells. 60-64. (Canceled)
 65. A method of inhibiting the immuneresponse in animals which comprises the method of claim
 59. 66. A methodof inhibiting organ rejection in animals receiving transplant organswhich comprises the method of claim
 65. 67. (Canceled)
 68. A method ofinhibiting autoimmune responses in animals suffering from an idiopathicautoimmune disease comprising the method of claim
 65. 69. The method ofclaim 68, wherein the idiopathic autoimmune disease comprises psoriasis,rheumatoid arthritis, Myasthenia gravis, systemic lupus erythematosus,Graves' disease, idiopathic thrombocytopenia purpura, hemolytic anemiaor diabetes mellitus. 70-111. (Canceled)
 112. A method of killing tumorcells or neoplastic cells which express a protein which is specificallyrecognized by monoclonal antibody 5c8 produced by the hybridoma havingATCC Accession No. HB 10916 in an animal which comprises administeringto the animal an effective amount of the pharmaceutical composition ofclaim 16 wherein the monoclonal antibody is conjugated to a therapeuticagent to inhibit the proliferation of the tumor cells or neoplasticcells. 113-144. (Canceled)